The invention relates to identification of bio-active molecules from a combinatorial library of oligopeptides attached to solid phase supports. The peptides attached to a single bead have essentially the same amino acid sequence. The synthesis history of each peptide bead may be recorded on each solid support in a code of inert molecular tags, such that beads of interest can be rapidly and efficiently decoded. A photocleavable crosslinker allows release of some of the oligopeptide by exposure to UV light. Molecular tags if present, remain covalently bound to the beads for post-assay analysis. The bioactive molecules may be screened in cytotoxic T lymphocyte screening assays.
Cellular immunotherapy is emerging as a technologically and intellectually compelling anti-cancer treatment. The generation of an immune response against tumors has been demonstrated in several animal models and has been inferred from reports of spontaneous tumor regression in man (Stotter and Lotze, 1990, Cancer Cells; 2:44-55). Cytotoxic T-lymphocyte (CTL) responses can be directed against antigens specifically presented by tumor cells, both in vivo and in vitro, without the need for prior knowledge of the molecular mechanism by which the tumor arose. In animal models, established tumors can be eradicated by the adoptive transfer of T-cells that are specifically immune to the malignant cells (Buen et al., Immunol. Today; 15:11-15). Techniques of adoptive T-cell therapy have recently been applied to the treatment of human viral disease, but the application of similar T-cell therapy for human malignancy has been hindered in part by the lack of well defined tumor antigens recognizable by autochthonous T-cells. Many human progressive or metastatic cancers, such as disseminated malignant melanoma or metastatic renal cell carcinoma, are resistant to conventional therapies, including chemotherapy and radiotherapy. In these types of cancers, immunotherapy has been tried over the past 10 years and although its success rate has been relatively modest, it remains a promising alternative to the conventional therapies (Bergmann et al., 1990, Onkologie; 13:137).
In man, spontaneous destruction of melanoma cells occurs in 15% to 20% of primary lesions, indicating that host protective mechanisms which can selectively destroy melanoma cells are present (Bystryn et al., 1993, Heme. Onc. Annals. 1:301). Vaccine immunotherapy with crude or partially purified melanoma vaccines can prevent tumor growth in 50% to 100% of mice immunized to otherwise lethal doses of melanoma cells. The protection is specific, indicating it is mediated by immune mechanisms. The challenge is to devise vaccine strategies that will induce similar immunoprotective responses in man.
For immunotherapy to be improved, epitopes recognized by tumor-specific-CTLs must be identified. CTL epitopes are 8-10 amino acid peptides derived from cellular proteins that are endocytically processed and presented on the tumor cell surface by major histocompatability complex (MHC) class I and class II glycoproteins. MHC molecules are expressed in virtually all nucleated cells and the combination of peptide and MHC molecule is specifically recognized by the appropriate T-cell receptors (TCRs). T-cells in the presence of antigen presenting cells and their corresponding antigen proliferate and acquire potent cytolytic activity.
Identification of the antigens recognized by these tumor-specific CTLs is vital to the rational development of peptide-based anti-tumor vaccines. A common strategy in the search for tumor antigens is to isolate tumor-specific T-cells and attempt to identify the antigens recognized by the T-cells. In patients with cancer, specific CTLs have been often derived from lymphocytic infiltrates present at the tumor site (Weidmann et al., 1994, xc2x0 Cancer Immunol. Immunother. 39:1-14). These tumor infiltrating lymphocytes (TILs) are a unique cell population that can be traced back to sites of disease when they are labeled with indium and adoptively transferred.
Indeed, the presence of a large number of T-cells in tumors has been correlated with a prognostically favorable outcome in some cases (Whiteside and Parmiani, 1994, Cancer Immunol. Immunother. 39:15-21). Recently it was shown that implantation of polyurethane sponges containing irradiated tumor cells can efficiently trap anti-tumor CTLs (4-times greater than lymph fluid, 50-times greater than spleen or peripheral blood) (Woolley et al., 1995, Immunology, 84: 55-63). Following activation with T-cell cytokines in the presence of their appropriately presented recognition antigen, TILs proliferate in culture and acquire potent anti-tumor cytolytic properties (Weidmann et. al., 1994, supra). Thus, TILs are a convenient source of lymphocytes greatly enriched for cells with rumor cell specificity. Additionally, tumor-specific CTLs have been found in peripheral blood or malignant ascites of patients with cancer, indicating that a systemic response to the tumor may be present or that redistribution of CTLs from the tumor to the periphery might occur (Wallace et al., 1993, Cancer Res. 53:2358-2367). In either case; this is an attractive feature for the, immunotherapeutic treatment of metastatic or disseminated cancers.
The reasons why tumor cells may express tumor-specific antigens (TSAs) are beginning to be understood. For example, TSAs may be the result of the processes of carcinogenesis, which are generally thought to stem from damage to a large number of genes, some of which have a role in the molecular mechanisms regulating cell growth and division. This damage results in uncontrolled cellular proliferation that defines the transformed cell. Thus, possible origins of TSAs include self proteins (such as fetal antigens) oncogene a products (including fusion proteins), mutated tumor suppressor gene products, other -mutated cellular proteins, or foreign proteins such as viral gene products. Nonmutated cellular proteins may also be antigenic if they are expressed aberrantly (e.g., in an inappropriate subcellular compartment) or in supernormal quantities. Given the numerous steps of cellular transformation and sometimes bizarre genotypes observed in cancer cells, it could be argued that tumor cells are likely to contain many new antigens potentially recognizable by the immune system.
Reports of shared tumor antigens are frequent in the literature. In the case of melanoma, there is recent evidence that the same T-cell-defined tumor antigens are expressed by independent human melanoma suggesting that transformation-associated events may give rise to recurrent expression of the same tumor antigen in different tumors of related tissue and cellular origin (Sahasrabudhe et al., 1993, J. Immunol., 151:6302-6310; Shamamian et al., 1994, Cancer Immunol. Immunother., 39:73-83; Cox et al., 1994, Science 264:716; Peoples et al., 1993, J. Immunol., 151:5481-5491; Jerome et al., 1991, Cancer Res., 51:2908-2916; Morioke et al., 1994, J. Immunol., 153:5650-5658). Previous studies in animal models have, in contrast, suggested that most chemical and ultraviolet radiation-induced tumors are antigenically diverse and that tumor rejection antigen may be generated by random mutation (Srivastava et al., 1986, Proc. Natl. Acad. Sci. USA, 83:3407-3411). However, it is highly improbable that a completely random process would give rise to shared antigens even in very closely related tumors. This data supports the possibility that specific anti-tumor immunotherapies, such as vaccines, may be active against more than one form of cancer and that the same vaccine may be effective against independently derived tumors of the same type.
While isolation, expansion, and retransfusion of TILs is appealing, there are severe adverse cardiorespiratory and hemodynamic effects such as tachycardia, increases in cardiac index, systemic vascular resistance, and pulmonary artery diastolic pressure which appear within two hours post-infusion. These effects are similar to the physiologic changes seen in interleuken-2 (IL-2) therapy and septic shock (Marincola et al., 1993, J. Immunol., 13:282-288). These changes are sustained and augmented by subsequent IL-2 administration (Lee et al., 1989, J. Clin. Oncol., 7:7-20). IL-2 is a T-cell cytokine and its production is among the earliest events following stimulation of the T-cell receptor (TCR). The physiological changes observed in septic shock have been associated with elevated levels of TNF-xcex1 and IL-6, both of which are produced upon T-cell stimulation (Calandra et al., 1990; J. Infect. Dis., 161:982-987).
A comprehensive survey of the literature reveals that neither adoptive transfer of tumor-specific CTLs nor specific active immunotherapy with whole tumor cells or cell-derived preparations leads to eradication of tumors or long term survival in more than a minority of patients. It has been demonstrated in vitro that peptides have succeeded in priming T-cells where cell-derived preparations have failed (Cox et al.,.1994, supra). Peptides that are expressed by the tumors of many individuals may be useful for immunotherapy, but the most generally applicable would be those that also are recognized by lymphocytes obtained from a large number of different cancer patients. Epitopes recognized by multiple CTL lines would be promising candidates for use in peptide-based anti-tumor vaccines. In the absence of a reliable iterative method to identify TSAs, there is no way of assessing the limits of cross-reactivity.
There are several obstacles which contribute to the difficulty of analyzing MHC-associated peptides by classical means. Current protocols involve isolating and assaying extremely pure MHC molecules from antigen presenting cells. Prior to peptide extraction, all contaminating proteinaceous material must be removed (this includes low molecular weight contaminants that normally escape detection by routine methods used to analyze protein purity such as SDS-PAGE) (Chicz and Urban, 1994, Immunol. Today, 15:155-160).
Briefly, immunoaffinity purification yields approximately 0.5-1 mg of HLA molecules per gram (11 culture) of B-cell lymphocytes (yields from B-cells are significantly higher than those obtained from primary explant tissues). Since the bound peptide is only 8-10 amino acids long. 1 mg of MHC contains 16 pmol of extractable peptide. Furthermore, the efficiency of peptide extraction is typically 75-80%. Thus, 1 mg of MHC usually yields 13 pmol of isolated peptide for analysis. The population of bound peptide is estimated to have a complexity  greater than 2000, the majority of which are believed to be self-peptides. Therefore, the average molar amount of each individual peptide present after purification is 13 pmol divided by the population complexity. The utility of a large pool of purified peptides in which each individual species is present in exceedingly minute quantities is limited. At this point, the purified peptides can be fractionated by HPLC and the fractions assayed for reactivity with cloned CTLs. Tandem mass spectrometry can be used to sequence reactive fractions. However, the complexity of peptides in each fraction often exceeds the number of peptides that can be sequenced with the available material. Thus, although this method has been used successfully, the lack of data in the literature gleaned from this approach is testimony to the difficulty of its successful execution.
Knowledge of the primary sequence of MHC, or of known T cell epitopes, has not yielded a key to immunogenicity of such epitopes. Identification and screening of epitopes has also not been further facilitated by the determination of structural features of the MHC, e.g., using X-ray crystallography. These techniques, which in other systems provide for the rational design or identification of receptor agonists and antagonists, have not proven useful for identification of T cell epitopes.
Recombinant bacteriophage have been used to produce large libraries. Using the xe2x80x9cphage methodxe2x80x9d (Scott and Smith, 1990, Science 249:386-390; Cwirla, et al., 1990, Proc. Natl. Acad. Sci., 87:6378-6382; Devlin et al., 1990, Science, 249:404-406), very large libraries can be constructed (106-108 chemical entities). However, in these libraries it is difficult to dissociate a response due to the recombinant fusion protein from one due only to the peptide. Another approach uses primarily chemical methods, of which the Geysen method (Geysen et al., 1986, Molecular Immunology 23:709-715; Geysen et al. 1987, J. Immunologic Method 102:259-274) and the recent method of Fodor et al. (1991, Science 251, 767-773) are examples. Furka et al. (1988, 14th International Congress of Biochemistry, Volume 5, Abstract FR:013; Furka, 1991, Int. J. Peptide Protein Res. 37:487-493). Houghton (U.S. Pat. No. 4,631,211, issued December 1986) and Rutter et al. (U.S. Pat. No. 5,010,175, issued Apr. 23, 1991) describe methods to produce a mixture of peptides that can be tested, as agonists or antagonists. However, these methods are deficient as they provide either limited numbers of predesigned peptides, which require some advance predictions about the desired sequence, or in a large (and in the case of Rutter, chaotic and indiscriminate) mixture of peptides, leaving one no better off than with naturally purified MHC containing peptide epitopes.
A major advance in screening occurred with the development of synthetic libraries (Needels et al., 1993, xe2x80x9cGeneration and screening of an oligonucleotide encoded synthetic peptide library,xe2x80x9d Proc. Natl. Acad. Sci. USA 90:10700-4; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Lam et al., International Patent Publication No. WO 92/00252, each of which is incorporated herein by reference in its entirety), and the like can be used to screen for receptor ligands.
The synthesis of indexed combinatorial peptide displays has been described (Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA, 90:10922-26). It is possible to synthesize epitope-length peptides on Merrifield resin beads while cosynthesized inert molecular tags allow rapid and efficient decoding of the synthesis history of any unique bead via gas-phase chromatography (Ohlmeyer et al., supra) The efficiency of decoding is about 90 percent a utilizing a single bead. Furthermore, it is not necessary to restrict assays to solid-phase interactions since photocleavable linkages allow controlled release of required amounts of peptide for solution-phase assays. This is important because it may not be possible for peptides to bind directly to surface-localized MHC class I molecules directly (in general, loading APCs with antigen occurs by internalizing the peptide and combining it with the MHC molecule as it assembles), and even if the APCs can bind directly to the beads, tight packing of the peptide on the surface of the beads may cause enough stearic hindrance so as not to allow access of the MHC/peptide complex to the CTL T-cell receptors. Note that MHC/peptide complexes have a remarkable stability. A feature of most MHC/peptide complexes is their unusually slow dissociation kinetics, with a half-life in the range of several days. Most peptides ( greater than 90% of characterized human HLA-A epitopes) will bind with affinities of 2-50 nM.
Van der Zee (1989, Eur. J. Immunol., 19:43-47) and coworkers have developed a powerful but limited strategy for identifying T-cell epitopes. Briefly, utilizing the xe2x80x9cpepscanxe2x80x9d technique, they were able to simultaneously synthesize several dozens of peptides on polyethylene rods arrayed in a 96-well microtiter plate pattern. This is similar to an indexed library in that the position of each pin defines the synthesis history on it. Peptides were then chemically cleaved from the solid support and supplied to irradiated syngeneic thymocytes for antigen presentation. The cloned CTL line was then tested for reactivity in a proliferation assay monitored by 3H-thymidine incorporation. This type of analysis particularly suits a CTL stimulation assay since it can be automated using a microtiter plate reader and employs relatively low levels of radiation. The procedure successfully identified a reactive epitope in a defined region of a 65 kDa mycobacterial heat shock protein with essentially no background. A second screen where the synthesized peptides had one alanine insertion per peptide at each position of the naturally occurring epitope identified an additional seven peptides with diminished yet detectable reactivity, underscoring the tolerances to substitutions in this assay. Additionally, screening peptides having a single deletion per peptide (derived from the natural epitope) yielded no reactive peptides, underscoring the specificity endowed by the presence of the nine residues in the naturally occurring epitope.
Notwithstanding the efforts made to date to identify T cell epitopes, the inventor herein has recognized a clear need in the art for a rapid method to obtain saturating profiles of epitopes which elicit CTL cytolytic activity directed against appropriate APCs. In several cases, derivatized natural epitopes are more effective than the natural epitope itself, accordingly, there is a need to identify such derivatized natural epitopes. Additionally, identification of epitopes from a wide range of independently derived CTLs will allow the design of powerful vaccines which are cross-reactive against different diseases and thus serve a greater cross-section of the population.
The present invention is directed to the design of degenerate, oligopeptide libraries comprising MHC allele-specific agretopes displaying diverse T cell receptor (TCR) epitopes, and a method of use of these libraries to screen for TCR epitopes. In particular, the present invention provides a method for designing oligopeptide libraries such that all species contain high affinity MHC allele-specific binding sites and a repertoire of variable domains that will interact with complementary TCRs. MHC allele-specific anchor residues have been determined for the common human MHC haplotypes.
For example, in a specific embodiment, a completely degenerate octamer library would have a complexity of 2.56xc3x971010. Two fixed anchor positions reduces the complexity to 6.4xc3x97107. The present invention incorporates knowledge of TCR promiscuity with respect to tolerance to conservative substitutions in naturally derived epitopes, combined with knowledge of MHC agretopes, to identify reactive epitopes and all reactive epitope derivatives. Thus, the present invention enables production of a library that can be practicably screened and whose signal to noise ratio is experimentally-tolerable.
The present invention further provides a method for screening an oligopeptide library for bioactive CTL epitopes such that pooled aliquots of solid phase supports can be simultaneously assayed with multiple CTL clones of the same MHC restriction class.
In a broad aspect, the present invention is directed to a method for identifying a cytotoxic T cell epitope. According to the invention, the method comprises the steps in order of contacting a population of at least two cytotoxic T cell (CTLs) having the same MHC-haplotype restriction with a library of molecules attached to solid phase supports by a releasable linker, wherein each solid phase support is attached to a single species of molecule, and wherein the structure of the molecule can be determined. The library of molecules contains a conserved structural motif corresponding to a structural motif characteristic of peptides that associate with the MHC-haplotype to which the cytotoxic T cells are restricted; this motif is referred to herein and in the art as an xe2x80x9cagretope.xe2x80x9d The library is contacted or exposed to antigen presentation means prior to or simultaneously with the CTLs, which antigen presentation means correspond to the MHC-haplotype to which the cytotoxic T cells are restricted. The solid phase supports of the library are in separate fractions, so as to facilitate identification of molecules that prove to be epitopes recognized by the CTLs. At least a portion of the releasable linker is cleaved so as to release at least a portion of the molecule, and the cytotoxic T cells are evaluated as to whether they recognize a molecule present in one or more of the fractions of the library of molecules. Upon observation of such recognition, the method then involves isolating one or more solid phase support from the fractions and determining the structure of a molecule on a solid phase support isolated from the fraction. In a specific embodiment, such molecules are peptides. However, the term peptide is construed herein to encompass peptidomimetics, and non-naturally occurring peptide analogs, as these have been developed in the art. It should be further recognized by those of skill in the art that molecules can be designed by empirical techniques, such as the combinatorial libraries described herein, that topologically and functionally perform as a peptide epitope, but which bear no structural resemblance to peptides (such as morphine activates opioid receptors but has a vastly different structurexe2x80x94except at the receptor-binding surfacexe2x80x94than xcex2-endorphin).
In a preferred aspect, the cytotoxic T cells are polyclonal T cells isolated from a site of cytotoxic T cell infiltration from an individual. Alternatively, such cells may be isolated from a site of cytotoxic T cell infiltration from two or more individuals, which two or more individuals share an MHC haplotype. In another embodiment, the CTLs may be two or more cytotoxic T cell lines. In yet another embodiment, the CTLs may be any combination of the foregoing.
In a further preferred aspect, the site of cytotoxic T cell infiltration is a tumor. The tumors from which cells or cell lines are obtained can be the same type of tumor in different individuals with a shared MHC haplotype, e.g., a melanoma from Mr. A and a melanoma from Ms. B, or different types of tumors from different individuals who share an MHC haplotype, e.g., a melanoma from Mr. A and a breast cancer from Ms. B.
Alternatively, CTL infiltrates can be from sites of viral infection, autoimmune inflammation (such as demyelinated nerve tissue or cerebrospinal fluid in MS, inflamed joints in arthritis, etc.), transplantation rejection, and like sites of inflammation or lymphocyte/leukocyte infiltration.
As noted above, the peptide identified according to the invention may comprise subunits selected from the group consisting of glycine, L-amino acids, D-amino acids, non-classical amino acids, and peptidomimetics.
Various solid phase supports (also termed herein xe2x80x9cbeadsxe2x80x9d) can be used in the practice of the invention. Such solid phase supports must be compatible with the biological assay to be performed, and must be inert to the synthesis of the molecule, e.g., peptide, and if present, a coding molecule. Examples of solid phase supports include polystyrene resin, poly(dimethylacryl)amide-grafted styrene-co-divinylbenzene resin, polyamide resin, polystyrene resin grafted with polyethylene glycol, and polydimethylacrylamide resin.
The releasable linker may release upon exposure to an acid, a base, a nucleophile, an electrophile, light, an oxidizing agent, a reducing agent, or an enzyme.
The invention provides specific structural motifs (agretopes) for use in libraries of the invention, including, but not limited to, LXXXXXXV (SEQ ID NO:1); RXXXXXX+(SEQ ID NO:2); X(D,E)XXXXXX(F, K,Y) (SEQ ID NO:3): RXXXXXXL (SEQ ID NO:4); X(K,R)XXXXX(L,I) (SEQ ID NO:5); (M,L)XXXXXXK (SEQ ID NO:6); EXXXXXX(Y,F) (SEQ ID NO:7); XPXXXXX(F,H,W,Y) (SEQ ID NO:8); (L,I)XXXXX(H,K) (SEQ ID NO:9); wherein X indicates any amino acid residue, and +indicates a positively charged amino acid.
In a preferred aspect, the invention greatly simplifies a primary search for an epitope by incorporating a limited number of representative amino acid residues in the peptides of the library. For example, positively charged amino acid residues may be substituted with an amino acid selected from the group consisting of lysine, arginine, and histidine; negatively charged amino acid residues may be substituted with an amino acid selected from the group consisting of aspartic acid and glutamic acid; neutral, polar amino acid residues may be substituted with an amino acid selected from the group consisting of asparagine, glutamine, serine, threonine, tyrosine, glycine, and cysteine; nonpolar amino acid residues may be substituted with an amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine. In a further embodiment, the nonpolar, aromatic amino acid residues are substituted with an amino acid selected from the group consisting of tyrosine, threonine, and tryptophan; and the nonpolar aliphatic amino acid residues are substituted with an amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, proline, and methionine.
The structure of the molecule (or peptide) determined in a simplified primary screen can be refined in a secondary screen. This secondary screening constrains the structure of the library of molecules to have the require agretope, and to have a sequence of chemically similar residues as defined in the primary screen. However, in the secondary screen all possible amino acids corresponding to a particular residue type are tested to find an epitope with the greatest stimulatory activity. Thus, the foregoing method may further comprise the steps of contacting at least one of the CTLs in the population of at least two cytotoxic T cells having the same MHC-haplotype restrictions with a library of molecules as set forth above, which library of molecules contains a conserved structural motif corresponding to a structural motif characteristic of peptides that associate with the MHC-haplotype to which the cytotoxic T cells are restricted, and wherein every amino acid corresponding to the representative residue is utilized at the position identified for the corresponding representative residue. The library is contacted or exposed to antigen presentation means prior to or simultaneously with the CTLs, which antigen presentation means correspond to the MHC-haplotype to which the cytotoxic T cells are restricted; at least a portion of the releasable linker is cleaved so as to release at least a portion of the molecule; the cytotoxic T cells are evaluated for recognition of a molecule present in one or more of the fractions of the library of molecules; one or more solid phase support from the fractions is isolated; and the structure of a molecule on a solid phase support from the fraction is determined.
In a specific embodiment, the invention provides a method for identifying a high affinity cytotoxic T cell epitope comprising contacting a population of cytotoxic T cells having an MHC-haplotype restriction with a library of molecules attached to solid phase supports by a releasable linker, wherein each solid phase support is attached to a single species of molecule, and wherein the structure of the molecule can be determined, which library of molecules contains a conserved structural motif corresponding to a structural motif characteristic of peptides that associate with the MHC-haplotype to which the cytotoxic T cells are restricted, and wherein every amino acid corresponding to a representative residue determined as set forth above is utilized at the position identified for the corresponding representative residue; and antigen presentation means, which antigen presentation means correspond to the MHC-haplotype to which the cytotoxic T cells are restricted; wherein the solid phase supports of the library are in separate fractions; cleaving at least a portion of the releasable linker so as to release at least a portion of the molecule; evaluating whether the cytotoxic T cells recognize a molecule present in one or more of the fractions of the library of molecules; isolating one or more solid phase support from the fractions; and determining the structure of a molecule on a solid phase support isolated from the fraction.
In a further preferred aspect of the invention, a coding molecule is attached to each solid phase support of the library, which coding molecule defines the structure of the molecule attached to the solid phase support by the releasable linker. In specific embodiments, the coding molecule is a peptide, an oligonucleotide, or, preferably, an inert molecular tag that can be decoded by gas-phase chromatography. An example of the latter is a halogen substituted benzene.
Thus, in one aspect, the structure of the molecule is determined by analyzing a portion of the molecule remaining on the solid phase support. For example, a sequence of the peptide may be determined by sequencing a portion of the peptide remaining on the solid phase support, e.g., using Edman degradation and microsequencing techniques. Alternatively, using the coding molecule technology set forth above, the structure of the molecule is determined by analyzing the structure of the coding molecule. In a further embodiment, the structure of the molecule is determined after isolating more than one candidate solid phase support; repeating the screening procedure with the isolated candidate supports, however, testing each support in a separate assay, isolating one such solid phase support that demonstrates CTL activation, and determining the structure of a molecule on that solid phase support.
According to the invention, the antigen presentation means may be a purified MHC class I molecule complexed to xcex22-microglobulin; an intact antigen presenting cell; or a foster antigen presenting cell. Preferably, the antigen presentation means is a foster antigen presenting cell. More preferably, the foster antigen presenting cell lacks antigen processing activity, whereby it expresses MHC molecules free of bound peptides. In a specific embodiment, the foster antigen presenting cell is the human B and T lymphoblast hybrid cell line 174xCEM.T2, deposited as a publicly available cell line with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, and assigned ATCC accession number CRL-1992.
The recognition of a molecule present in one or more of the fractions of the library of molecules by the cytotoxic T cells can be evaluated by detecting cytotoxic T cell activation. For example, but not by way of limitation, cytotoxic T cell activation can be detected by a method selected from the group consisting of 3H-thymidine incorporation; metabolic activity detected by conversion of MTT to formazan blue; increased cytokine mRNA expression; increased cytokine protein production; increased protein synthesis; and chromium release by target cells.
In another aspect, the invention provides a method of identifying a protein antigen comprising identifying the cytotoxic T cell epitope of the protein determined in a secondary screen (or refinement screen), comparing a sequence of the T cell epitope identified in step (a) with known sequences of proteins; and determining a protein having a sequence corresponding to the sequence of the T cell epitope. It should be recognized that although the secondary screen of the invention may provide a highly active epitope that does not correspond to the natural epitope, and thus may not provide sequence identity, in all likelihood the sequence of the natural epitope will correspond to a portion of the sequence of the antigen, or be so similar as to leave little doubt about the antigen (Blake et al., 1996, J. Exp. Med. 184:121-30).
It should also be recognized that an epitope derived according to the present invention may correspond to an as yet unidentified protein. Thus, the invention provides for identification of novel protein antigens. Furthermore, the invention provides a method for cloning the cDNA and genomic DNA encoding such protein by generating degenerate oligonucleotide probes or primers based on the sequence of the epitope.
The present invention further provides therapeutic and diagnostic agents comprising oligopeptide sequences determined according to the foregoing methods.
Diagnostic agents may also include oligonucleotides corresponding to the identified epitope or a region of genomic DNA surrounding the epitope locus. For example, in the case of the CDK4 epitope discussed infra, PCR was used to type individuals from the patient""s pedigree for the presence of the CDK4 mutation, thus identifying individuals at risk for developing this melanoma.
Accordingly, it is a primary object of the present invention to provide a method to rapidly and efficiently identify CTL epitopes.
It is a particular object to identify such CTL epitopes of tumor antigens, and more particularly ubiquitous epitopes found on a wide variety of tumors.
It is another object to identify CTL epitopes of other disease-associated antigens, such as but not limited to viral antigens, autoimmune antigens, and the like.
Still another object of the invention is to prepare a vaccine comprising an epitope or epitopes identified according to the invention for protection from tumors or other diseases, including viral infections, autoimmune disease, and the like.